In fermentation and biotransformation processes the metabolic activity of pro- and/or eukaryotic cells is used to produce substances such as beer, wine and bio-ethanol, L-glutamic-, citric- and lactic acid, different antibiotics, enzymes, steroids and aroma ingredients.
Products obtained by fermentation or biotransformation may contain DNA from the cells used in the process. On the one hand purification of intracellular products requires cell disruption upon which intracellular compounds including DNA are liberated into the broth and even after several further purification steps residual DNA may remain in the product. On the other hand also extracellular products may contain residual DNA even though an efficient separation step for cell removal is used after fermentation or biotransformation—just by cell lysis and liberation of DNA during the fermentation or biotransformation process.
For products obtained by fermentation or biotransformation and still containing DNA the regulatory requirements may be stricter than for those which do not contain residual DNA. E. g. for food and feed products obtained by fermentation of genetically modified microorganisms (GMMs) absence of GMMs and newly introduced genes in the product has a direct impact on its categorization for risk assessment purposes (EFSA (2011) The EFSA Journal 9(6): 2193). Absence of newly introduced genes should be shown by PCR spanning the full length of the coding sequence(s) of the target gene(s) of concern (EFSA (2011) The EFSA Journal 9(6): 2193).
Hence, from a regulatory point of view a reliable and cost efficient method for DNA fragmentation resulting in the absence of complete genes from the product may be of special interest.
Several approaches to achieve DNA fragmentation are known and described in the scientific literature, including enzymatic or chemical DNA degradation (Anderson (1981) Nucleic Acids Res. 9: 3015-3027; Roe (2004) Methods Mol. Biol. 255: 171-187; Bauer et al. (2003) Eur. Food Res. Technol. 217: 338-343; Poirier (2004) Nature Rev. Cancer 4: 630-637), shear forces (hydrodynamic shearing (Joneja and Huang (2009) Biotechniques 46: 553-556; Thorstenson et al. (1998) Genome Res. 8: 848-855), sonication (Deininger (1983) Anal. Biochem. 135: 247-263), nebulization (Burger et al. (2007) Nat. Protocols 2: 603-614)), oxidative attack (Aronovitch et al. (1991) Free Radic. Res. Commun. 12-13: 499-508), irradiation (Rastogi et al. (2010) Journal of Nucleic Acids; Yang and Hang (2013) J. Biomol. Tech. 24:98-103) and radicals (Dizdaroglu and Jaruga (2012) Free Radic. Res. 46:382-419).
However, for some of these methods such as the application of shear forces additional equipment is required which makes the overall process more complex and expensive. Further, when using chemical or enzymatic DNA fragmentation the enzyme or chemical compound has to be removed after the fragmentation step.
Hence, there is still a need for a simple method for removing DNA from biotechnologically produced products which does not impact product yield and product quality.
The present invention provides a method for removing DNA from biotechnologically produced products which reliably results in the absence of complete genes in the product while minimizing product losses.